Production of adenovirus vectors with reduced levels of replication competent adenovirus contamination

Abstract

Methods, cells and recombinant adenoviral vectors are disclosed that permit the production of recombinant adenoviral vector stocks with reduced levels of contamination by replication competent adenoviruses (RCA). In certain embodiments are disclosed early region 1 (E1) deficient recombinant adenoviral vectors and complementing E1 positive host cells whose sequences are designed to avoid formation of RCA by homologous recombination between sequences in the vector and E1 sequences in the cells. One aspect of the invention involves the inversion of the packaging signal in a recombinant adenoviral vector relative to an adjacent or nearby inverted terminal repeat (ITR). Methods include use of site-specific intregrase family recombinases such as Cre or FLP and recombinase recognition sites such as lox sites or frt sites.

1. A recombinant adenoviral nucleic acid sequence comprising an adonoviral vector comprising: a segment of DNA comprising an adenoviral packaging signal, the segment providing the packaging signal in an inverted orientation with respect to the nearest of a left adenoviral ITR and a right adenoviral ITR of the vector; a deletion in early region 1 (E1); and an insertion of foreign DNA in E1, wherein the packaging signal is functional and wherein the insertion of foreign DNA in E1 comprises an expression cassette. 
     
     
       2. The recombinant adenoviral nucleic acid sequence according to  claim 1 , wherein the packaging signal is positioned within about 600 nucleotides from the left adenoviral ITR. 
     
     
       3. The recombinant adenoviral nucleic acid sequence of  claim 1 , wherein the packaging signal is positioned within about 600 nucleotides from the right adenoviral ITR. 
     
     
       4. The recombinant adenoviral nucleic acid sequence of  claim 1 , wherein the insertion of foreign DNA comprises a cloning site comprising at least one site, each of said at least one site is selected from the group consisting of an endonuclease site and an integrase family recombinase target site. 
     
     
       5. A recombinant adenoviral nucleic acid sequence comprising an adenoviral vector comprising:
 a. a left-end adenoviral inverted terminal repeat (ITR) sequence; 
 b. an adenoviral packaging signal inverted with respect to its normal orientation in relation to the nearest of the left-end adenoviral ITR and a right-end adenoviral inverted terminal repeat (ITR) sequence; 
 c. a deletion in early region 1 (E1) to eliminate expression of E1A and E1B genes; 
 d. an insertion of foreign DNA in E1, or in both E1 and E3, wherein foreign DNA inserted into E1, or each of the foreign DNA insertions in E1 and E3, comprises an expression cassette; and 
 e. the right-end adenoviral inverted terminal repeat (ITR) sequence. 
 
     
     
       6. A recombinant adenoviral vector comprising an adenoviral packaging signal in an inverted orientation with respect to the nearest of a right and a left adenoviral inverted terminal repeat (ITR) sequence, and further comprising an insertion into early region 1 (E1) of foreign DNA comprising an expression cassette comprising an introduced DNA sequence, a promoter operatively linked to the introduced DNA sequence, and a termination sequence. 
     
     
       7. The recombinant adenoviral vector of  claim 6 , additionally comprising an insertion of foreign DNA in early region 3 (E3). 
     
     
       8. The recombinant adenoviral vector of  claim 6  wherein the inverted packaging signal is disposed at the right end of the adenovirus DNA sequence adjacent to the right adenoviral ITR. 
     
     
       9. A system for propagation of a recombinant adenoviral vector comprising
 a. a culture vessel comprising culture media into which are added: 
 b. a recombinant adenoviral vector comprising a nucleic acid sequence comprising:
 i. a left-end adenoviral inverted terminal repeat (ITR) sequence; 
 ii. a deletion in early region 1 (E1) to eliminate expression of E1A and E1B genes; 
 iii. an insertion of an expression cassette in E1, or in both E1 and E3; 
 iv. an adenoviral packaging signal inverted with respect to a normal orientation in relation to the nearest of the left-end adenoviral ITR and a right-end adenoviral inverted terminal repeat (ITR) sequence; and 
 v. the right-end adenoviral inverted terminal repeat (ITR) sequence, and 
 
 c. a cell comprising an adenoviral DNA sequence expressing E1A and E1B genes. 
 
     
     
       10. The system of  claim 9 , the adenoviral DNA sequence of the cell lacking a left adenoviral ITR. 
     
     
       11. A method for producing a recombinant adenoviral vector comprising a gene of interest, said method not producing a vector having a functional E1 region, said method comprising:
 a. providing a complementing cell harboring a first nucleic acid comprising adenoviral nucleic acid sequences encoding functional E1A protein and functional E1B protein; 
 b. transferring a second nucleic acid into said complementing cell, said second nucleic acid comprising a recombinant adenoviral nucleic acid sequence comprising functional adenoviral Inverted Terminal Repeats (ITRs) at or near both of its termini, and a functional adenoviral packaging signal inverted with respect to a normal orientation along the adenoviral DNA sequence with respect to the nearest of the adenoviral ITRs, the second nucleic acid further compirising a foreign DNA sequence in early region 1 (E1) comprising the gene of interest, and all sequences required for replication of said second nucleic acid which are not provided by said complementing cell; 
 c. culturing said complementing cell; and 
 d. harvesting the recombinant adenoviral vector produced from the complementing cell. 
 
     
     
       12. The method of  claim 11 , wherein the first nucleic acid lacks a left adenoviral ITR.